Myosin is a family of proteins that demonstrate force-generated ATPase activity when it interacts with actin. Myosin heavy chain (MHC) protein isoform diversity in vertebrates is generated by multiple genes as well as by alternative splicing of the pre-mRNA. Previous studies in this laboratory have demonstrated the existence of a neuron-specific nonmuscle MHC-B isoform, in addition to the ubiquitously expressed form of nonmuscle MHC-B. This neuron-specific isoform is generated by alternative splicing of a single cassette type of exon N30 which is located between constitutive exons E5 and E6. Inclusion or exclusion of exon N30 is cell type-specific, with inclusion being restricted to neuronal cells and being regulated during cell differentiation. To understand the mechanism(s) of neuron-specific alternative splicing, we have been characterizing cis-elements which are required for neuron- specific recognition of the N30 exon using a minigene transfection system. Deletion and mutation analysis of minigenes demonstrates that the following three cis-elements are required for regulated splicing of N30: 1) an intronic distal downstream enhancer (IDDE) located 1.5 kb downstream from the N30 exon; 2) an exonic enhancer present in the N30 exon; and 3) the suboptimal sequence of the polypyrimidine tract preceding the N30 exon. We have also examined the effects of the IDDE on the in vitro splicing of the pre-mRNAs using neuronal nuclear extracts. The presence of the IDDE causes enhancement of the splicing of the pre-mRNA consisting of the constitutive exon E5 and the alternative exon N30. In contrast, pre-mRNA consisting of the N30 and the constitutive exon E6 are efficiently spliced with and without the IDDE. These results suggest that the IDDE functions via the 3 splice site preceding the regulated N30 exon. - alternative RNA splicing; neuron-specific; nonmuscle myosin heavy chain-B gene